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  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
  • C12P7/56—Lactic acid
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12M23/00—Constructional details, e.g. recesses, hinges
  • C12M23/58—Reaction vessels connected in series or in parallel
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
  • C12M29/18—External loop; Means for reintroduction of fermented biomass or liquid percolate
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
  • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
  • C12M41/18—Heat exchange systems, e.g. heat jackets or outer envelopes
  • C12M41/22—Heat exchange systems, e.g. heat jackets or outer envelopes in contact with the bioreactor walls
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
  • C12M41/26—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pH
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
  • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
  • C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor

Definitions

• the present invention is in the field of a method of producing lactic acid in high yield in a sequencing batch reactor.
• glucose or other sugars may be used as feedstock for bacteria, which ferment the glucose into lactic acid.
• the reactor is at least partly operated under defined conditions and in a cyclic mode.
• Lactic acid (CH 3 CH (OH) CO 2 H) is a simple organic chemical compound that can be used in many applications. It is a chiral molecule and it occurs as L- or D-lactic acid. It is noted that lactic acid is highly soluble.
• Lactic acid fermentation is performed on an industrial scale by lactic acid bacteria (Lactobacillus species) , which convert simple carbohydrates such as glucose, sucrose, or galactose to lactic acid, or by chemical synthesis. Lactic acid producing bacteria can produce two moles of lactate from one mole of glucose, or one mole of lactate from one mole of glucose as well as carbon dioxide and acetic acid/ethanol, which latter is not preferred.
• lactic acid bacteria Lactic acid bacteria
• Lactic acid producing bacteria can produce two moles of lactate from one mole of glucose, or one mole of lactate from one mole of glucose as well as carbon dioxide and acetic acid/ethanol, which latter is not preferred.
• lactic acid fermentation is performed under rather strict conditions.
• a relatively pure culture such as Lactobacillus delbrueckii.
• the aqueous solution in which fermentation is performed is typically partly neutralized, such as with lime.
• a pH of about 4.5-5.0 is used.
• the fermentation temperature is >50°C as production is often negligible up to 45°C.
• a fermentor and seed reactor are used.
• the yield of lactic acid is rather high, such as > 0.9 lactic acid/g glucose (yield on carbon of 90%), the productivity is good (e.g. >5 g/l*h) and the titer relatively high (>150 g/1 lactic acid) .
• a pure culture may attribute to some 15% of production costs.
• the inoculum costs some 3%, and energy consumption some 4%.
• the fermentor is relatively large (a few thousand cubic meter) , the seed reactor also has a significant volume (a few hundred cubic meters), and the need to be made from a relatively expensive material, such as stainless steel, making
• Lactic acid is a precursor for polylactic acid (PLA) , which can be atactic or syndiotactic, and which polymers are biodegradable polyesters. Lactic acid may also be used as a food conservative, in cosmetics, and as a pharmaceutical component. The market size for lactic acid is huge (>5
• the present invention therefore relates to an improved method of producing lactic acid by fermentation, which solves one or more of the above problems and drawbacks of the prior art, providing reliable results, without jeopardizing
• the present invention relates to a method of producing lactic acid in a sequencing batch reactor
• adaptively cycling at least once between (i) a reaction phase, (ii) an effluent phase, and (iii) a feed phase, preferably 2-5000 times adaptive cycling, more preferably 4-1000, such as 5-1000 times.
• Each phase is considered to relate to a period of time.
• an adaptive cycle can be ended almost immediately when completed, consumption of substrate (e.g. glycose) is as fast as possible, resulting in high time-averaged volumetric rates.
• substrate e.g. glycose
• 1-20 adaptive cycles per day are possible, such as 2-10 adaptive cycles.
• Operation of the reactor can be continued for long periods of time. Maintenance can be performed 1-4 times per year, or less.
• the present method uses a mixed culture capable of fermenting saccharide into lactic acid, which is added, such as to the reactor. Compared to prior art methods as described above the present method is considered to be >20% cheaper in terms of unit production costs of lactic acid.
• the present method provides a high start biomass enabling a high productivity, such as > 6 g/l*h, the possibility of reusing biomass as protein and vitamin source therewith reducing peptide and vitamin source consumption, the (indirect) production of CO2 is reduced significantly, scheduled down-time is low as no sterilization is required, and unscheduled down time, such as due to phage infection, is very unlikely due to the mixed culture
• the biomass may be recycled, especially as it may be still intact, and therewith contributing to an increase in productivity and yield.
• the throughput is higher.
• the down side is that somewhat lower yields (70% versus 90%) are obtained, which may be due to more biomass and possible byproduct, and in general there is no direct control over D/L ratio lactic acid; however, under specific conditions the latter can be resolved.
• D- lactic acid ⁇ 0.1%) is formed and > 99.9% L-lactic acid.
• the present method comprises a reaction phase wherein (ial) maintaining the pH at a predetermined level between 5.6 and 8.5, preferably between 5.9 and 8.0, more preferably between 6.4 and 7.5, such as at 7.0, which is compared to prior art methods relatively high, (ia2) maintaining the temperature at 30-80 °C, preferably at 32-72 °C, more
• the stationary phase can be determined adequately, albeit with a small delay in time.
• (ic2) When fermentation has reached the stationary phase removing part of the effluent to the effluent phase, and (ic3) adding feed to the reaction phase, therewith largely or fully replenishing the reaction phase.
• the feed can be added subsequently to removing effluent from the reaction phase, during reaction, before reaction, and combinations thereof.
• the effluent phase at least part of the lactic acid is removed, and (iib) preferably a remainder of the effluent phase is provided to the feed phase, preferably wherein 30-70% of broth is provided to the feed phase, such as 40-60% of broth.
• the lactic acid may be remover through an outlet towards a vessel or the like, it may be precipitated in the reactor, such as by adding Ca 2+ , it may be separated over a membrane, and combinations
• a part of the broth may be recycled.
• lactic acid may be removed from the reaction phase, such as by in-situ separation.
• the feed phase (iiia) an aqueous feed mixture is provided
• saccharide comprising >10 g/1 of a saccharide comprising compound, wherein the saccharide compound is selected from glucose, sucrose, fructose, galactose, lactose, disaccharides, oligo saccharides, poly saccharides, such as with 3-100
• saccharide comprising compound is a single compound, such as solely glucose, and >1 g peptide/100 g saccharide compound, or a combination thereof, wherein the peptide concentration in the feed phase is between 1-30 g peptide/100 g saccharide. It has been found that in addition to a saccharide as basic feed compound also peptides are required in order to obtain sufficient yield. Without the amount of peptide mentioned the yield drops to some 10%, much lower than the present 70% typically obtained.
• a biomass hydraulic retention time is controlled between 4h-144h, such as between 1-8 days. It is noted that the solid retention time and hydraulic retention time may be substantially the same, especially when continuous mixing is applied during volume exchange, such as during effluent removal.
• the HRT is considered to depend on cycling time, which is controlled by the adaptive cycling.
• the present invention relates to an apparatus for performing a method according to the invention, which may be a relatively cheap apparatus, such as a plastic or concrete apparatus, comprising a feed container, the feed container having a volume of 1-50 m 3 , the feed container being in fluidic contact with a reactor over a feed valve, the reactor having a pH-sensor in fluidic contact with the aqueous solution of the reactor, a reservoir comprising a base and a reservoir valve and optionally a pump, the reactor having a volume of 10-100 m 3 , the reactor being in fluidic contact with an effluent container over an effluent valve, the effluent container having a volume of 1-50 m 3 , and the effluent container being in fluidic contact with the feed container, each container and reactor having a mixer for rotating an aqueous phase with 1-600 rpm, the effluent container having an outlet for removing lactic acid, and the feed container having an input for replenishing feed, and a controller adapted (a)
• the present invention provides a solution to one or more of the above-mentioned problems and overcomes drawbacks of the prior art .
• biomass may be adaptive cycled at least once between the reaction phase, the effluent phase, and the feed phase. It has been found that biomass not only contributes to fermentation of saccharide into lactic acid, but also provides peptides, therewith improving the yield and throughput .
• the feed phase may comprise > 80 g/1 saccharide compound, such as 100- 200 g/1.
• the peptide concentration in the feed phase is between 1-40 g peptide/100 g saccharide, preferably between 2-30 g/100 g, more preferably between 5-15 g/100 g.
• the peptide may be selected from monopeptides, dipeptides, tripeptides, tetrapeptides, or is provided as microbial biomass, and combinations thereof.
• biomass also peptides in yeast extract, or whey, or by-product of biological origin may be used.
• biomass in the present system may be recycled, providing peptides, as well as vitamins or precursors thereof.
• a reactor size may be 50-1000 m 3 , such as 100-300 m 3 .
• Such is much smaller than prior art reactors, therewith significantly reducing energy consumption in the production process, such as die to agitation (stirring) of the reaction phase.
• the reactor may be a sequencing batch reactor or a sequencing fed batch rector.
• the present reactor may relate to a reactor comprising at least one of a zone of an apparatus, a phase of operation of an apparatus, and a sub-reactor.
• the sequencing in combination with adaptive cycling provides a quick start up, high throughput, and good adaptability of the system to potential varying characteristics.
• no live yeast is present in the feed phase stock.
• the above relate to a significant reduction in costs of operation, simplified mode of operation, reduced down-time, and reduced risk of infections.
• the feed phase may comprise a vitamin B and/or metabolic precursor thereof, preferably 10 _2 -10 mg vitamin B/g saccharide and/or precursor, preferably 2*10 _2 -5 mg/g vitamin B and/or
• the vitamin B may be selected from vitamin Bi (thiamine) , vitamin B2 (riboflavin), vitamin B3 (niacin, nicotinamide, nicotinamide riboside) , vitamin Bs (pantothenic acid) , vitamin Bb (pyridoxine) , vitamin B7 (biotin) , vitamin B12 (cobalamin) , a salt thereof, such as a phosphate salt, and combinations thereof.
• vitamin Bi thiamine
• vitamin B2 riboflavin
• vitamin B3 niacin, nicotinamide, nicotinamide riboside
• vitamin Bs pantothenic acid
• vitamin Bb pyridoxine
• vitamin B7 biotin
• vitamin B12 cobalamin
• a salt thereof such as a phosphate salt, and combinations thereof.
• the precursor may be selected from metabolic precursors for coenzyme in catabolism of sugar, co-factor FAD, co-factor FMN, coenzyme NAD, coenzyme NADP, coenzyme A, a metabolic coenzyme, a fatty acid metabolism coenzyme, an amino acid metabolism coenzyme, and combinations thereof.
• a culture titer of lactic acid of >40 g/1 may be maintained, typically >50 g/1, such as > 100 g/1, that is high titers are obtainable .
• a magnesium (cation) concentration in the feed phase may be 0.1-5 g/1, such as 0.2-2 g/1.
• a calcium (cation) concentration in the feed phase may be >1.5 mg Ca/g saccharide. So, for 10-200 g saccharide/1 more than 1.5-300 mg Ca/L may be provided.
• the magnesium and calcium are found to contribute to fermentation.
• the mixed culture may be enriched, such as by increasing an amount of saccharide compound, e.g. to >100 g/1, such as > 200g/l .
• a biomass retention time may be controlled, such as between 1-8 days .
• the reaction phase comprises >10% Streptococcus, such as > 50% Streptococcus, which can be determined by a semi-quantitative method like fluorescent in situ hybridization, or with any other method, such as with PCR.
• a hydraulic retention time may be from 1-8 days
• a base may be selected from hydroxides, oxides, ammonia, and combinations thereof, preferably comprising Ca 2+ , Na + , or Mg 2+ , and combinations thereof.
• a pH may be maintained at 7.0D0.5, a temperature at 30-55 DC, a peptide amount at >2g/100g saccharide, a vitamin B at
• L-lactic acid is produced, such as > 99.9% L-lactic acid (on a mol lactate/mol saccharide comprising compound basis) .
• L-lactic acid on a mol lactate/mol saccharide comprising compound basis
• Figs, la-b, 2, and 3a-b and 4 show some details of the present invention.
• a feed phase is provided with a saccharide, and typically mixed.
• the feed is provided, in an adaptive cyclic mode, to a reaction phase, wherein
• microorganisms produce lactic acid from the saccharide.
• the pH is measured.
• a source of base is activated, and base is added until the pH reaches a predetermined level. Then the addition of base is stopped, for the time being.
• the effluent of the reaction phase is transferred to an effluent phase, and the lactic acid is removed. A remainder of the effluent phase is fed back to the feed phase.
• Figure 2 Result of a FISH image using the str probe (light grey) , specific for the streptococcus genus (Trebesius et al . 2000) compared to the EUB338 probe mix (dark grey) (Amann et al . 1990) .
• Figure 4 Development of the read abundance of the 16S rRNA gene V3-V4 region in time on genus level. Various species are found, which develop over time in amount.
• Streptococcus, Enterobacteriaceae, Citrobacter, Klebsiella, and Clostridium are found in relatively high amount.
• anti-foaming agent 3% v:v anti-foam C, Sigma Aldrich, Germany was added in equal amounts and at equal speed as NaOH during 10 g L -1 glucose fermentations.
• the enrichment at 10 g L -1 glucose was performed using a medium to which NH 4 CI, KH 2 PO 4 and FeCl 2 .4H 2 0 were added to obtain a set molar C:N:P:Fe ratio of 100:5:1:0.33 and 1.5 g yeast extract was added per 10 g of glucose.
• Glucose and yeast extract solutions were autoclaved separately at 110°C for 20 minutes and then combined. Salts were supplied to the reactor from a second vessel, which was autoclaved at 121°C. Magnesium concentrations were adjusted to increasing glucose concentrations. Trace elements were supplied in sufficient amounts .
• the reactor was operated in SBR mode. In a start-up phase for culture development the reactor was operated in batch mode until glucose was entirely consumed.
• 10 mL (0.5% v/v) of suspended and sieved (150 pm filtered) soil from the botanical garden of TU Delft was used (pH 7.4) and 10 mL of anaerobic digester sludge
• VSS volatile suspended solids
• the batch reactor was operated as described for the batch process at pH 5.
• the reactor was inoculated with cell pellets of approximately 1L of the culture obtained at the end of the enrichment at pH 7. Samples were taken every 30 minutes for the first 3.5 hours and every hour until glucose was nearly depleted ( ⁇ 16 mM residual glucose) . Anti-foam was added manually when foaming occurred. Culture was stored in the fridge overnight before collecting the pellet for VSS
• DNA was extracted from cell pellets from different time points in the enrichment using the DNAeasy microbial
• Fluorescent in situ hybridization was used for analyzing the microbial community with epifluorescence
• Streptococcus was observed to be predominant genus, with also several Enterobacteriaceae genera occurring, such as Klebsiella and Citrobacter. The amount of Streptococcus was shown to be very dominant, in the range of >90% of the biomass .
• bacteria could be successfully enriched at both pH 5 and pH 7 using the described enrichment strategy. Further, lactic acid production at pH 7 is favoured over pH 5, as the productivity is higher and there is selective production of L-lactic acid.

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